Traditional use of Cissampelos pareira L. for hormone disorder and fever provides molecular links of ESR1 modulation to viral inhibition

Bioactive fractions or compounds obtained from medicinal plants have been used for the treatment of multiple diseases. This effect could be due to common pathways underlying these conditions that are targeted by such medicines. In this study, we explored the molecular basis of action of one such herbal formulation Cissampelos pareira, used for the treatment of female hormone disorders and fever. Genome-wide expression studies on MCF7 cell lines treated with Cipa extract were carried out using Affymetrix arrays. Transcriptome analysis revealed a downregulation of signatures of estrogen response governed by estrogen receptor (ER). Molecular docking analysis identified 38 constituent molecules in Cipa that potentially bind ({Delta}G< -7.5) with ER at the same site as estrogen. Cipa transcriptome signatures show high positive connectivity (https://clue.io/) scores with protein translation inhibitors such as emetine (score: 99.61) and knockdown signatures of genes linked to the antiviral response such as ribosomal protein RPL7 (score: 99.92), which is also an ER coactivator. Cipa exhibits antiviral activity in dengue infected MCF7 cells that is decreased upon ESR1 (estrogen receptor 1) gene knockdown. This approach reveals a novel pathway involving ESR1-RPL7 axis that could be a potential target in dengue viral infection.

Rapid, accurate, nucleobase detection using FnCas9

Rapid detection of pathogenic sequences or variants in DNA and RNA through a point-of-care diagnostic approach is valuable for accelerated clinical prognosis as has been witnessed during the recent COVID-19 outbreak. Traditional methods relying on qPCR or sequencing are difficult to implement in settings with limited resources necessitating the development of accurate alternative testing strategies that perform robustly. Here, we present FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that employs a direct Cas9 based enzymatic readout for detecting nucleotide sequences and identifying nucleobase identity without the requirement of trans-cleavage activity of reporter molecules. We demonstrate that FELUDA is 100% accurate in detecting single nucleotide variants (SNVs) including heterozygous carriers of a mutation and present a simple design strategy in the form of a web-tool, JATAYU, for its implementation. FELUDA is semi quantitative, can be adapted to multiple signal detection platforms and can be quickly designed and deployed for versatile applications such as infectious disease outbreaks like COVID-19. Using a lateral flow readout within 1h, FELUDA shows 100% sensitivity and 97% specificity across all range of viral loads in clinical samples. In combination with RT-RPA and a smartphone application True Outcome Predicted via Strip Evaluation (TOPSE), we present a prototype for FELUDA for CoV-2 detection at home. Single sentence summaryA method to identify nucleotide sequence or nucleobase identity using FnCas9 and its implementation in the rapid and accurate diagnosis of SARS-CoV-2

High throughput detection and genetic epidemiology of SARS-CoV-2 using COVIDSeq next generation sequencing

The rapid emergence of coronavirus disease 2019 (COVID-19) as a global pandemic affecting millions of individuals globally has necessitated sensitive and high-throughput approaches for the diagnosis, surveillance and for determining the genetic epidemiology of SARS-CoV-2. In the present study, we used the COVIDSeq protocol, which involves multiplex-PCR, barcoding and sequencing of samples for high-throughput detection and deciphering the genetic epidemiology of SARS-CoV-2. We used the approach on 752 clinical samples in duplicates, amounting to a total of 1536 samples which could be sequenced on a single S4 sequencing flow cell on NovaSeq 6000. Our analysis suggests a high concordance between technical duplicates and a high concordance of detection of SARS-CoV-2 between the COVIDSeq as well as RT-PCR approaches. An in-depth analysis revealed a total of six samples in which COVIDSeq detected SARS-CoV-2 in high confidence which were negative in RT-PCR. Additionally, the assay could detect SARS-CoV-2 in 21 samples and 16 samples which were classified inconclusive and pan-sarbeco positive respectively suggesting that COVIDSeq could be used as a confirmatory test. The sequencing approach also enabled insights into the evolution and genetic epidemiology of the SARS-CoV-2 samples. The samples were classified into a total of 3 clades. This study reports two lineages B.1.112 and B.1.99 for the first time in India. This study also revealed 1,143 unique single nucleotide variants and added a total of 73 novel variants identified for the first time. To the best of our knowledge, this is the first report of the COVIDSeq approach for detection and genetic epidemiology of SARS-CoV-2. Our analysis suggests that COVIDSeq could be a potential high sensitivity assay for detection of SARS-CoV-2, with an additional advantage of enabling genetic epidemiology of SARS-CoV-2.